The present invention relates to genomic DNA and plasmid DNA of aphids Buchnera sp.
Buchnera sp. APS is a bacterial symbiont harbored by aphids. The host aphids are insects belonging to the suborder homoptero of the order hemiptera. Nearly 10,000 species of them are known throughout the world. Aphids have extremely strong fertility based on diploid parthenogenesis, and are one of the most serious agricultural insect pests on the earth. Aphids harbor many bacteria called Buchnera sp. in specialized cells, called bacteriocyte. The mutualism between Buchnera and aphids is so obligate that the symbiont Buchnera cannot survive outside the host aphid and aphids lacking Buchnera lose their fertility in addition to decreased growth.
Hence, noticing the host-symbiont relationship of the aphid and Buchnera is useful to obtain information to destroy aphids.
The present invention is to provide genomic DNA and plasmid DNA of Buchnera sp.
The present inventors have succeeded in determining a whole nucleotide sequence of genome of Buchnera, which is a bacterial symbiont harbored by Acyrthosiphon pisum and in identifying 619 genes (including plasmids) contained in the genome as a result of diligent research on the above problems.
That is, the present invention provides genes derived from Buchnera sp., comprising DNA of (a) or (b) as follows.
(a) a DNA selected from a group consisting of a DNA having a nucleotide sequence ranging from a start point to an end point as shown in Table 1 in a nucleotide sequence represented by SEQ ID NO:1, or a DNA complementary thereto, and
(b) a DNA hybridizing to said DNA of (a) under stringent conditions and encoding a protein having a function same as that of the product expressed by the DNA.
Here, the term xe2x80x9cthe product expressed by said DNAxe2x80x9d means one of (a substance encoded by a sequence ranging from a start point to an end point) substances described in xe2x80x9cSubstance Namexe2x80x9d column of Table 1.
Further, the present invention provides a recombinant vector containing the above gene or a transformant containing the vector.
Furthermore, the present invention provides genomic DNA of Buchnera sp. having a nucleotide sequence represented by SEQ ID NO:1.
Furthermore, the present invention provides a plasmid derived from Buchnera sp., comprising DNA of the (c) or (d) as follows.
(c) a DNA having a nucleotide sequence represented by SEQ ID NO:2 or 3, and
(d) a plasmid, capable of hybridizing to the DNA having a nucleotide sequence represented by SEQ ID NO:2 or 3 under stringent conditions, and self-replicating.
Further, the present invention provides a method of producing the above-mentioned protein, comprising the steps of culturing the transformant and collecting the protein expressed by a target gene from the resulting culture product.
Hereinafter, a more detailed explanation of this invention will be given. The present specification includes the contents of the specifications and/or drawings of the Japanese Patent Applications No. 2000-107160 based on which the present application claims priority.
The present invention relates to genomic DNA with a length of approximately 640 kb of Buchnera sp. (hereinafter also referred to as Buchnera) and two plasmid DNAs present in Buchnera sp.
1. Cloning of Buchnera genomic DNA and plasmids
Buchnera can be obtained by the following techniques. For example, the host aphids harboring Buchnera are dissected, and huge cells (called bacteriocyte) in which Buchnera is living are isolated. The bacteriocytes are crushed and filtered through a 5 xcexcm pore size filter, thereby isolating Buchnera. Buchnera can also be isolated by homogenizing aphids and filtering the homogenates through 20, 10, and 5 xcexcm pore size filters in order. Moreover, Buchnera can be isolated by density gradient centrifugation using sucrose or percoll (Pharmacia).
An example of aphids is Acyrthosiphon pisum (Harris).
Next, genomic DNA is prepared from Buchnera. The genomic DNA can be prepared by known methods including a phenol/chloroform protocol.
Thus obtained DNA can be analyzed by the whole genome shotgun sequencing in this invention. The whole genome shotgun sequencing is to provide information on a whole genomic sequence, comprising the steps of fragmenting and sequencing randomly the whole genome in large quantities, and searching fragment ends overlapping to each other using a computer to join them together. That is, this method involves sequencing each DNA fragment treated with restriction enzymes or each DNA fragment fragmented at a random site using HydroShear (GeneMachines) and the like, comparing the sequences to each other to find overlapping portions, and then connecting the overlapping ends of the fragments, whereby determining the whole sequence.
This technique is basically the same as that of Fleischmann R. D. et al (Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 469-512, 1995). In order to avoid chimera formation in preparing shotgun sequence libraries, some methods (for example, Partial Fill-in method) can be adapted. In the partial fill-in method, bases of overhang ends are partially polymerized.
The nucleotide sequences of the above DNA fragments can be determined by known techniques including Sanger method (Molecular Cloning, vol. 2, 13.3, 1989) and methods based on PCR. Normally, nucleotide sequences are determined by performing sequencing reactions with PRISM sequencing kit and the like containing fluorescent dideoxy terminator (Perkin Elmer), and using an autosequencer (model ABI 377, Applied Biosystem).
SEQ ID NO:1 represents the whole sequence of the genomic DNA of this invention. In addition, Table 1 shows all the genes (608 genes excluding plasmids) contained in the nucleotide sequence of the chromosome represented by SEQ ID NO:1. 572 genes encoding proteins contained in the above genes can be isolated by, for example, PCR method. In Table 1, xe2x80x9cFxe2x80x9d represents + chain and xe2x80x9cRxe2x80x9d represents xe2x88x92 chain in the data in xe2x80x9cOrientationxe2x80x9d column. xe2x80x9cTypexe2x80x9d represents the sequence type of a gene. For example, CDS represents translation regions for proteins, tRNA transfer RNA, rRNA ribosomal RNA, and PS pseudogenes. Pseudogenes (PS) contain frameshift mutation or a stop codon inserted in the middle. When a direction is xe2x80x9cF,xe2x80x9d data in xe2x80x9cStart pointxe2x80x9d column represents an initiation point for translation of a substance to be encoded by the gene, and data in xe2x80x9cEnd pointxe2x80x9d column represents a termination point for the translation. For example, in Table 1, a second (BU002) gene (gene name: atpB) corresponds to a nucleotide sequence from 2278th to 3102nd bases and encodes ATP-synthetase A-chain. When a direction is xe2x80x9cR,xe2x80x9d translation proceeds in the direction opposite to that of the complementary strand from an initiation to an end point. For example, in Table 1, a 10th (BU010) gene (gene name: gyrB) represents a complementary strand of a nucleotide sequence from 8911th to 11322nd bases of a nucleotide sequence of SEQ ID NO:1. Translation proceeds in the direction from 11322nd to 8911th base based on the sequence position in SEQ ID NO:1. The remainder genes also encode substances (proteins, enzymes nucleic acids and the like) described in xe2x80x9cSubstance namexe2x80x9d column according to nucleotide sequences between xe2x80x9cStart pointxe2x80x9d and xe2x80x9cEnd pointxe2x80x9d described in Table 1 or their complementary sequences.
Next, each nucleotide sequence or its complementary sequence of the genes located between start points to end points of Table 1 is determined. Once the sequence has been determined, each of the genes can be obtained by chemical synthesis, by PCR using a nucleotide sequence at 5xe2x80x2 or 3xe2x80x2 end of the gene as a primer and using the whole or a part of genomic DNA (SEQ ID NO:1) as a template, or by hybridization using a nucleotide sequence of the gene described in Table 1 or DNA fragment having its complementary sequence thereof as a probe.
The genes of the present invention also include a gene hybridizing to the above-mentioned DNA under stringent conditions and encoding a protein having the same function as that of a product (a substance encoded by a sequence from xe2x80x9cStart pointxe2x80x9d to xe2x80x9cEnd pointxe2x80x9d in Table 1) expressed by the DNA.
The term xe2x80x9cstringent conditionsxe2x80x9d means conditions by which specific hybrids are produced and non-specific hybrids are not produced. That is, DNAs that share high homology (60% or more homology, preferably 80% or more homology) hybridize to each other in such conditions. More specifically, sodium concentration ranges from 150 to 900 mM, preferably 600 to 900 mM, and temperature ranges from 60 to 68xc2x0 C., preferably 65xc2x0 C.
In addition to the above-described genomic DNA, plasmids can also be isolated from Buchnera sp. in this invention.
Plasmids of this invention can be prepared in the same manner as for genomic DNA. Nucleotide sequences of the plasmids of this invention are also determined simultaneously with the genomic chromosome by the above-mentioned shotgun sequencing.
Two types of the plasmids, pLeu and pTrp, are obtained as described above, each containing a self-replication sequence derived from Buchnera sp. The plasmids have the following sequences and possess features as shown in Table 2. Table 2 shows 11 genes contained in nucleotide sequences of the plasmids represented by SEQ ID NOS: 2 and 3.
pLeu (leucine plasmid): SEQ ID NO:2
pTrp (tryptophan plasmid): SEQ ID NO:3
In Table 2, each column of xe2x80x9cOrientation,xe2x80x9d xe2x80x9cType,xe2x80x9d xe2x80x9cStart point,xe2x80x9d and xe2x80x9cEnd pointxe2x80x9d represent the same as described in Table 1.
The plasmids of the present invention also include those containing DNA, capable of hybridizing to DNA comprising a nucleotide sequence of SEQ ID NO: 2 or 3 under stringent conditions, and self-replicating, in addition to those containing DNA comprising a nucleotide sequence of SEQ ID NO:2 or 3. The term xe2x80x9cstringent conditionsxe2x80x9d can be defined as described above.
2. Construction of a recombinant vector and a transformant
Recombinant vectors of this invention can be obtained by ligating the above gene to an appropriate vector. A transformant can be obtained by introducing the recombinant vector of this invention into a host so that a gene of interest can be expressed.
Examples of vectors include phages or plasmids, which can autonomously replicate in host microorganisms. Examples of plasmid DNA include plasmids derived from Escherichia coli (for example, pBR322, pBR325, pUC118, pUC119, pUC18, and pUC19), plasmids derived from Bacillus subtilis (for example, pUB110, and pTP5), plasmids derived from yeast (for example, YEp13, YEp24, and YCp50). Examples of phage DNA include xcex phage (Charon4A, Charon21A, EMBL3, EMBL4, xcexgt10, xcexgt11, and xcexZAP). Further, examples of vectors also include animal viruses, such as retro virus and vaccinia virus, and insect viruses, such as baculo virus.
To insert the gene of this invention into a vector, for example, purified DNA is cleaved with an appropriate restriction enzyme and inserted to a restriction enzyme site or multicloning site of an appropriate vector DNA so as to ligate to the vector.
The gene of this invention must be incorporated into a vector in order to exhibit its function. A promoter and the gene of this invention can be ligated to the vector of this invention. If necessary, a cis element, such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) can also be integrated to the vector. Examples of selection markers include dihydrofolic acid reducing enzyme gene, ampicillin-resistant gene, neomycin-resistant gene. In addition to vectors capable of replicating autonomously in two or more types of host microorganisms, such as Escherichia coli and Bacillus brevis, various shuttle vectors can be used. Fragments of the vectors can also be obtained by cleaving with the above-mentioned restriction enzymes.
To ligate a DNA fragment to a vector fragment, a known DNA ligase is used. After annealing, a DNA fragment is ligated to a vector fragment so as to construct a recombinant vector.
Hosts used for transformation are not specifically limited so far as they can express the gene of this invention. Examples of the host cells include bacteria belonging to the genera Escherichia, such as Escherichia coli, the genera Bacillus, such as Bacillus subtilis, and the genera Pseudomonas, such as Pseudomonas putida, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells, such as COS and CHO cells, and insect cells, such as Sf9.
When a bacterium such as Escherichia coli is used as a host cell, a preferable recombinant vector can autonomously replicate in the bacterium and comprises a promoter, a ribosome binding sequence, the gene of this invention, and a transcription termination sequence. The recombinant vector may also contain a gene to regulate a promoter.
Examples of Escherichia bacteria include, E. coli DH5xcex1 and Bacillus bacteria include Bacillus subtilis, but not limited thereto.
Any promoter that can be expressed in a host cell may be used. Examples of such a promoter include promoters derived from Escherichia coli or phages, such as trp promoter, lac promoter, PL promoter, and PR promoter. Artificially designed and modified promoters, such as tac promoter may also be used.
Any method to introduce recombinant vectors into bacteria, that is, to introduce DNA into bacteria may be used and is not specifically limited. Examples of such methods include a method using calcium ion, electroporation and the like.
When yeast is used as a host cell, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. In this case, promoters used herein are not specifically limited so far as they can express in yeast. Examples of such a promoter include gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFxcex11 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and AOX1 promoter.
Methods to introduce recombinant vectors into yeast are not specifically limited. Any method to introduce DNA into yeast may be used. Examples of such methods include electroporation (Becker, D. M. et al., Methods. Enzymol., 194: 182, 1990), spheroplast method (Hinnen, A. et al., Proc. Natl. Acad. Sci., USA, 75, 1929, 1978), lithium acetate method (Itoh, H., J. B acteriol., 153, 163, 1983) and the like.
When an animal cell is used as a host cell, examples of host cells include mouse cells COS-7, Vero, Chinese hamster ovarian cells (CHO cells), mouse L cells, rat GH3, and human FL cells. Examples of promoters include SRxcex1 promoter, SV40 promoter, LTR promoter, and CMV promoter. In addition, an initial gene promoter of human cytomegalovirus may also be used. Examples of methods of introducing recombinant vectors into animal cells include electroporation, calcium phosphate transfection and lipofection.
When an insect cell is used as a host cell, Sf9 cells and the like are used. Examples of methods of introducing recombinant vectors into insect cells include calcium phosphate transfection, lipofection, and electroporation.
3. Production of useful substances
A whole or a part of the genes of the present invention, or a whole genomic DNA can be used as basic data for DNA analysis based on a simple metabolic system of Buchnera. For example, analysis made on function of genomic DNA having a nucleotide sequence of SEQ ID NO:1 or function of at least one gene out of genes shown in Table 1 provides genetic information involving the metabolic system. Such genetic information can be used for development of pesticides, which can suppress the growth of Buchnera by inhibiting specifically a part of the metabolic pathway of Buchnera.
Though aphids feed on plant sieve tube fluid, which is deficient in nutrients other than sugar, they have extremely strong fertility. This is because Buchnera supply nutrients (including essential amino acids, vitamin B and other unknown nutrients), which aphids cannot synthesize. Accordingly, the genomic data of Buchnera should contain useful genes encoding the above nutrients. That is, useful substances can be produced by expressing these genes.
Proteins of interest (useful substances) can be obtained in this invention by culturing the aforementioned transformants containing genes of interest and collecting the protein from the culture products. Here the term xe2x80x9cculture productxe2x80x9d means either culture supernatants, or culture cells or culture bacteria, or disrupted cells or bacteria.
The transformants of this invention are cultured in/on media by normal techniques employed for culturing hosts.
A medium for culturing transformants obtained by using microorganisms including Escherichia coli, yeast and the like as hosts contains a carbon source, a nitrogen source, and inorganic salts, which the microorganisms can assimilate, and allows the transformant to grow efficiently. Either natural media or synthetic media can be used if they satisfy the above conditions.
Examples of carbon sources include glucose, fructose, sucrose, and carbohydrates e.g., starch, organic acids e.g., acetic acid and propionic acid, and alcohol e.g., ethanol and propanol. Examples of nitrogen sources include ammonia, salts of inorganic acids or organic acids, e.g., ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, and corn steep liquor. Examples of inorganic substances include potassium primary phosphate, potassium secondary phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
Culturing is performed by shaking culture or submerged aeration-agitation culture under aerobic conditions at 37xc2x0 C. for 6 to 24 hours. The pH is kept within a range from 7.0 to 7.5 while culturing. The pH is adjusted using inorganic or organic acid, alkaline solutions or the like.
If necessary, an antibiotics e.g., ampicillin or tetracycline may be added to the media while culturing.
When microorganisms transferred with the expression vectors using inducible promoters are cultured, inducers may be added to the media if necessary. For example, isopropyl-xcex2-D-thiogalactopyranoside (IPTG) or the like may be added to the media when microorganisms transferred with the expression vectors containing lac promoter are cultured; indoleacrylic acid (IAA) or the like may be added when microorganisms transferred with the expression vectors containing trp promoter are cultured.
The media for culturing transformants obtained by using animal cells as host cells include generally used RPMI1640 media, DMEM media, or those to which fetal calf serum or the like is added. Normally, the transformant is cultured in the presence of 5% CO2 for 1 to 30 days at 37xc2x0 C. If necessary, antibiotics e.g., kanamycin and penicillin may be added to the medium while culturing.
When the protein of interest is produced within a bacterium or a cell, the protein is extracted by disrupting the bacterium or the cell. Further, when the protein of interest is produced outside a bacterium or extracellularly, the culture solution is used as it is or the bacterium or the cell is removed by centrifugation. Then the protein of interest can be isolated and purified from the aforementioned culture product by using appropriate combination of one or more of general biochemical techniques for isolation and purification of proteins, including ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography.
Whether the protein of interest is obtained or not can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.
SEQ ID NO:4 Synthetic DNA
SEQ ID NO:5 Synthetic DNA
SEQ ID NO:6 Synthetic DNA
SEQ ID NO:7 Synthetic DNA